Composition for modulating intestinal permeability and/or treating and/or preventing leaky gut related diseases, and method for modulating intestinal permeability and/or treating and/or preventing leaky gut related diseases

ABSTRACT

A composition for modulating intestinal permeability and/or treating and/or preventing leaky gut related diseases including a Chinese herbal compound material or a Chinese herbal compound extract is provided. The Chinese herbal compound material includes Ganoderma, red jujube, longan and lotus seed. Moreover, the Chinese herbal compound extract includes a Ganoderma extract, a red jujube extract, a longan extract and a lotus seed extract.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 62/981,720, filed on Feb. 26, 2020, the entirety of which isincorporated by reference herein.

This application claims priority of Taiwan Patent Application No.109146258, filed on Dec. 25, 2020, the entirety of which is incorporatedby reference herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

A sequence listing submitted as a text file via EFS-Web is incorporatedherein by reference. The text file containing the sequence listing isnamed “9044B-A27419-US_Seq_Listing.txt”; its date of creation was Dec.29, 2020; and its size is 2,188 bytes.

TECHNICAL FIELD

The present disclosure relates to a Chinese herbal compound material ora Chinese herbal compound extract, and particularly it relates to acomposition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases comprising a Chinese herbalcompound material or a Chinese herbal compound extract, and use of aChinese herbal compound material or a Chinese herbal compound extract.

BACKGROUND

The intestinal barrier regulates the stability of the intestinal tract.The main functions thereof include: (1) Water and ion transport: theintestinal tract can process about 9 liters of liquid per day, which ismainly absorbed by the small intestine, and fluid absorption andsecretion can be processed through transcellular or intercellularpathways. (2) Regulating antigens: reconciling antigens in theintestinal lumen is the key to maintaining proper immunity. Specificdendritic cells may be activated by changes in intercellularpermeability. (3) Immune defense: the intestinal lumen can assist immunedefense through the process of flushing microorganisms and toxins (theouter layer of the mucosa contains a large number of bacteria, while theinner layer maintains a sterile state).

The intestinal barrier is mainly composed of several layers of defensemechanism to limit the translocation of antigens in the intestinallumen. The intestinal barrier comprises a single layer of semipermeableepithelial cells, and the adherent and tight junctions proteins of theapical junctions thereof can join epithelial cells and regulate passingparacellular antigens and molecules through the epithelium. Intestinalepithelial cells transport antigens and molecules from the intestinallumen to the mucosa via transcellular pathways. Specific epithelialcells, such as M cells (Microfold cells, M cells) can transport antigensin the intestinal lumen to phagocytes and lymphocytes in the epitheliumto initiate an immune response. Goblet cells, Paneth cells andenterocytes can secrete mucins and antimicrobial peptides (AMPs) andaggregate into mucosa layer. In addition, the plasma cells of the laminapropria of the intestinal epithelium can secrete IgA. Intestinalepithelial cells also have many microbial recognition receptors (MRR),such as Toll-like receptors (TLRs) and NOD-like receptors, which canidentify specific microbial associated molecular patterns (MAMP). Theintestinal microbes identified by the intestinal epithelial cells caninduce the secretion of cytokines and other immunoregulatory factors,and can help to induce an immune regulatory response to combat theintestinal microbes and maintain the intestinal stability.

The intestinal barrier has the function of preventing the invasion ofpathogenic antigens and maintaining intestinal health, and theintestinal flora is an important component of the intestinal mucosalbarrier. When the intestinal flora maintains a balance, it can maintainnormal body functions. When the intestinal flora is out of balance dueto chronic stress, chronic constipation and exposure to environmentaltoxins (for example, taking antibiotics, unhealthy diet, etc.) to makemany bacteria be killed, the intestinal mucosa will be destroyed by badbacteria to cause food residues and toxins enter the bloodstream, andthis phenomenon is leaky gut, or increased intestinal permeability.Leaky gut or increased intestinal permeability can also trigger aninflammatory response.

Leaky gut can be caused by many factors, and can result in manydifferent clinical signs or diseases. Currently known intestinal barrierfunction abnormalities and intestinal flora imbalance related diseasesinclude inflammatory bowel disease (IBD), celiac disease (coeliacdisease), irritable bowel syndrome, acute pancreatitis, non-alcoholicfatty liver disease (NAFLD), alcoholic cirrhosis, type 1 and type 2diabetes (diabetes mellitus), obesity, chronic kidney disease,cardiovascular disease, multiple organ failure syndrome (shock, burnsand trauma), AIDS, asthma, eczema, psoriasis, autism, depression,anxiety, schizophrenia, bipolar disorder, Alzheimer's disease,Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis(ALS), ankylosing spondylitis, fibromyalgia, chronic sleepfragmentation, insomnia, etc.

However, so far no Chinese herbal medicine has been verified to be usedfor the treatment of leaky gut related diseases, and thus there is stillan urgent need for novel Chinese herbal medicines for the treatment ofleaky gut related diseases.

SUMMARY

The present disclosure provides a composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases, comprising: a Chinese herbal compound material or a Chineseherbal compound extract. The Chinese herbal compound material comprisesGanoderma, red jujube, longan, and lotus seed, and in the Chinese herbalcompound material, a weight ratio of Ganoderma, red jujube, longan andlotus seed is 0.1-15:0.6-2:0.6-2:0.6-5. Moreover, the Chinese herbalcompound extract comprises Ganoderma extract, red jujube extract, longanextract and lotus seed extract, and a weight ratio of Ganoderma, redjujube, longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theChinese herbal compound extract is 0.1-15:0.6-2:0.6-2:0.6-5.

The present disclosure also provides a method for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases, comprising administering a composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases to a subject in need thereof. The composition formodulating intestinal permeability and/or treating and/or preventingleaky gut related diseases, comprises: a Chinese herbal compoundmaterial or a Chinese herbal compound extract. The Chinese herbalcompound material comprises Ganoderma, red jujube, longan, and lotusseed. Moreover, the Chinese herbal compound extract comprises Ganodermaextract, red jujube extract, longan extract and lotus seed extract.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The present invention can be more fully understood by reading thesubsequent detailed description and examples with references made to theaccompanying drawings, wherein:

FIGS. 1A to 1P respectively show the results of the cell lethalconcentration test of the respective test samples prepared in Example 1;

FIG. 2 shows the results of the intestinal epithelial cell permeabilitytest of the single-ingredient extract. ***: p<0.001, compared to thenegative control group. The data are shown as mean±standard deviation(Mean±SD) (n=3). The statistical method is one-way ANOVA;

FIG. 3 shows the results of the intestinal epithelial cell permeabilitytest of the compound extract. Compared to the negative control group: *:p<0.05; ***: p<0.001. Compared to Sample 15A: #: p<0.05; ##: p<0.01;###: p<0.001. Compared to Sample 1: ϕ: p<0.05, ϕϕ: p<0.01, ϕϕϕ: p<0.001.The data are shown as mean±standard deviation (n=3). The statisticalmethod is one-way ANOVA;

FIGS. 4A to 4D show the results of the intestinal epithelial cellpermeability test of the four-ingredient compound extracts withdifferent proportions. Compared to the negative control group: *:p<0.05; **: p<0.01; ***: p<0.001. Compared to Sample 15A: #:p<0.05; ##:p<0.01; ###: p<0.001. Compared to Sample 1: ϕ: p<0.05, ϕϕ: p<0.01, ϕϕϕ:p<0.001. The data are shown as mean±standard deviation (n=3). Thestatistical method is one-way ANOVA;

FIG. 5 shows the results of the intestinal epithelial cell permeabilitytest of Sample 15B at different concentrations. Compared to the negativecontrol group treated with rhamnose: *: p<0.05; in the positive controlgroups and each experimental group, the group treated with rhamnolipidscompared to the group without rhamnose treatment: #: p<0.05. The dataare shown as mean±standard deviation (n=3). The statistical method isone-way ANOVA;

FIGS. 6A to 6C respectively show the effect of Sample 15B on theexpression of CLDN3, OCLN and TJP1 genes. The negative control group isuntreated cells, and the positive control group is treated withberberine chloride (BBC) at 50 μM (18.6 μg/mL). Compared to the negativecontrol group: *: p<0.05; **: p<0.01; ns: not significant. The data areshown as mean±standard deviation (n=3). The statistical method isone-way ANOVA;

FIG. 7 shows the effect of Sample 15B on the intestinal permeability ofDSS-induced mice. Compared to the untreated group: #: p<0.05. Comparedto the negative control group: *: p<0.05. The data are shown asmean±standard deviation (n=3-4). The statistical method is t test;

FIG. 8 shows the effect of Sample 15B on animals with circadiandisruption. Compared to the untreated group: ###: p<0.001. Compared tothe negative control group: ***: p<0.001. The data are shown asmean±standard deviation (n=6). The statistical method is that the datais one-way ANOVA and Dunnett's test;

FIGS. 9A, 9B and 9C respectively show the effect of Sample 15B on thetime spent in the holes, the frequency of hole-poking and theexploration activities of the rats in the evaluation test of locomotionand exploratory behavior. Compared to the Sham group: #: p<0.05, ##:p<0.01. Compared to the control group: *: p<0.05, **: p<0.01. The dataare shown as mean±standard deviation (n=6). The statistical method isthat the data is one-way ANOVA and Dunnett test;

FIG. 10 shows the effect of Sample 15B on the time for rats to find asafe platform in the swimming pool in a spatial performance in Morriswater maze test. Compared to the Sham group: #: p<0.05. Compared to thecontrol group: **: p<0.01; ***: p<0.001. The data are shown asmean±standard deviation (n=6). The statistical method is that the datais one-way ANOVA and Dunnett test;

FIG. 11 shows the effect of Sample 15B on the time for rats to reach thesafe platform in the swimming pool in a non-spatial performance inMorris water maze test. Compared to the Sham group: ###: p<0.001.Compared to the control group: *: p<0.05; ns: not significant. The dataare shown as mean±standard deviation (n=6). The statistical method isthat the data is one-way ANOVA and Dunnett test;

FIGS. 12A and 12B respectively show the effect of Sample 15B on the AChEactivity in the hippocampus area and frontal cortex area of rats in theanimal model of β-amyloid-induced learning impairment. Compared to theSham group: #: p<0.05, ###: p<0.001. Compared to the control group: **:p<0.01; ***: p<0.001; ns: no significant. The data is that statisticalmethod is that the data is one-way ANOVA and Dunnett test; and

FIG. 13 shows the results of high performance liquid chromatography(HPLC) analysis of Sample 15B.

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the disclosed embodiments. It will be apparent,however, that one or more embodiments may be practiced without thesespecific details.

The present disclosure may provide a composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases, which may comprise, but is not limited to, a Chineseherbal compound material or a Chinese herbal compound extract, but it isnot limited thereto.

A “leaky gut related disease” mentioned in the present disclosure is notparticularly limited, as long as it is a disease related to leaky gut.For example, a leaky gut related disease may comprise a disease of whicha symptom or which itself can be alleviated and/or treated and/orprevented by modulating intestinal permeability and/or by alleviatingand/or treating and/or preventing leaky gut, or a disease which isinvolved in an expression of a gene and/or protein related to leaky gut,and of which a symptom or which itself that can be alleviated and/ortreated and/or prevented by modulating the expression of the gene and/orprotein related to leaky gut, but it is not limited thereto. Example ofthe leaky gut related disease may comprise, but is not limited to,inflammatory bowel disease (such as colitis, etc.), celiac disease,irritable bowel syndrome, acute pancreatitis, non-alcoholicsteatohepatitis, alcoholic cirrhosis, type 1 diabetes, type 2 diabetes,obesity, chronic kidney disease, cardiovascular disease, multiple organfailure syndrome, AIDS, asthma, eczema, psoriasis, mental disease (suchas autism, depression, anxiety, schizophrenia, bipolar disorder, etc.),neurodegenerative disorder (such as Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, amyotrophic lateral sclerosis orcombinations thereof, etc.), ankylosing spondylitis, fibromyalgia, sleepdisorder (such as chronic sleep fragmentation, insomnia or a combinationthereof) or any combination thereof, etc.

In the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure mentioned above, the Chinese herbal compound materialmentioned above may comprise, but is not limited to Ganoderma, redjujube, longan, and lotus seed, and the Chinese herbal compound extractmentioned above may comprise, but is not limited to, Ganoderma extract,red jujube extract, longan extract and lotus seed extract. The Chineseherbal compound material mentioned above or the Chinese herbal compoundextract mentioned above has a modulating effect on intestinalpermeability and/or has a treating and/or preventing effect on leaky gutrelated diseases.

In the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure, the Ganoderma in the Chinese herbal compound materialmentioned above or the Ganoderma used as one of the preparation rawmaterials for the Chinese herbal compound extract mentioned above maycomprise Ganoderma lingzhi, Ganoderma sinensis, Ganoderma lucidum or anycombination thereof, but it is not limited thereto. In one embodiment,the Ganoderma in the Chinese herbal compound material mentioned above orthe Ganoderma used as one of the preparation raw materials for theChinese herbal compound extract mentioned above may be Ganodermalucidum.

In the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure, the red jujube in the Chinese herbal compound materialmentioned above or the red jujube used as one of the preparation rawmaterials for the Chinese herbal compound extract mentioned above maycomprise grey jujube, jixin jujube, winter jujube, big jujube, smalljujube, golden silk jujube or any combination thereof, but it is notlimited thereto. In one embodiment, the red jujube in the Chinese herbalcompound material mentioned above or the red jujube used as one of thepreparation raw materials for the Chinese herbal compound extractmentioned above may be grey jujube.

In the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure, the longan in the Chinese herbal compound material mentionedabove or the longan used as one of the preparation raw materials for theChinese herbal compound extract mentioned above may comprise, but is notlimited to, Fen Ke longan, Hong Ke longan, Qing Ke longan or anycombination thereof. In one embodiment, the longan in the Chinese herbalcompound material mentioned above or the used as one of the preparationraw materials for the Chinese herbal compound extract mentioned abovemay be Fen Ke longan.

Moreover, in the composition for modulating intestinal permeabilityand/or treating and/or preventing leaky gut related diseases of thepresent disclosure, the lotus seed in the Chinese herbal compoundmaterial mentioned above or the lotus seed used as one of thepreparation raw materials for the Chinese herbal compound extractmentioned above may comprise, but is not limited to, red lotus seed,white lotus seed or a combination thereof. In one embodiment, the lotusseed in the Chinese herbal compound material mentioned above or thelotus seed used as one of the preparation raw materials for the Chineseherbal compound extract mentioned above may be red lotus seed.

In one specific embodiment, the Ganoderma, red jujube, longan and lotusseed in the Chinese herbal compound material mentioned above or theGanoderma, red jujube, longan and lotus seed used as the preparation rawmaterials for the Chinese herbal compound extract mentioned above may beGanoderma lucidum, grey jujube, Fen Ke longan and red lotus seed,respectively.

In the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure, the composition per gram may at least contains about 0.2 mgof ganoderic acid A, for example, the composition per gram may at leastcontains about 0.2-20 mg of ganoderic acid A, such as about 0.2 mg,about 0.25 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.8 mg,about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20mg, etc., but it is not limited thereto. In one embodiment, the minimumcontent of the ganoderic acid A mentioned above can be used to confirmthe quality of the composition for modulating intestinal permeabilityand/or treating and/or preventing leaky gut related diseases of thepresent disclosure.

In one embodiment, in the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure, in the Chinese herbal compoundmaterial mentioned above, a weight ratio of Ganoderma, red jujube,longan and lotus seed may be about 0.1-15:0.6-2:0.6-2:0.6-5, such asabout 0.3-12:0.8-1.5:0.8-1.5:0.8-4, about 0.5-10:1:1:1-3, about 1:1:1:1,about 0.5:1:1:1, about 3:1:1:1, about 6:1:1:1, about 10:1:1:1, about1:1:1:3, but it is not limited thereto. In one specific embodiment, inthe composition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases of the present disclosure,in the Chinese herbal compound material mentioned above, a weight ratioof Ganoderma, red jujube, longan and lotus seed may be 1:1:1:1. Inanother specific embodiment, in the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure, in the Chinese herbalcompound material mentioned above, a weight ratio of Ganoderma, redjujube, longan and lotus seed may be 3:1:1:1.

In one embodiment, in the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure, the form of each herbal materialcontained in the Chinese herbal compound material mentioned above maycomprise original herbal material, slices/pieces obtained by cutting theoriginal herbal material, powder obtained by grinding the originalherbal material, etc., or any combination thereof, and it is notparticularly limited.

Furthermore, in one embodiment, the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure comprises the Chinese herbalcompound material mentioned above, and the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure can be packaged in a filterbag. In this embodiment, the filter bag packed with composition formodulating intestinal permeability and/or treating and/or preventingleaky gut related diseases of the present disclosure can be brewed witha solvent to obtain brewing liquid. In one specific embodiment, thefilter bag packed with composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure can be brewed with water to obtainbrewing liquid which can be taken directly.

Moreover, in one embodiment, in the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure, a weight ratio of Ganoderma,red jujube, longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theforegoing Chinese herbal compound extract may be about0.1-15:0.6-2:0.6-2:0.6-5, such as about 0.3-12:0.8-1.5:0.8-1.5:0.8-4,about 0.5-10:1:1:1-3, about 1:1:1:1, about 0.5:1:1:1, about 3:1:1:1,about 6:1:1:1, about 10:1:1:1, about 1:1:1:3, but it is not limitedthereto. In one specific embodiment, in the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure, a weight ratio of Ganoderma,red jujube, longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theforegoing Chinese herbal compound extract may be 1:1:1:1. In anotherspecific embodiment, in the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure, a weight ratio of Ganoderma, redjujube, longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theforegoing Chinese herbal compound extract may be 3:1:1:1.

Furthermore, in one embodiment, in the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure, in the foregoing Chineseherbal compound extract, a weight ratio of the Ganoderma extractmentioned above, the red jujube extract mentioned above, the longanextract mentioned above and the lotus seed extract mentioned above maybe about 1-40:20-200:20-180:5-130, such as about5-35:40-180:40-170:10-120, about 9.3:150.1:136.8:56.2, about5.3:172.28:156.37:64.25, about 6.18:100.5:91.21:112.45, but it is notlimited thereto.

The method for obtaining the Chinese herbal compound extract in thecomposition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases of the present disclosureis not particularly limited, as long as a mixture of all extracts of theherbal materials in the preparation raw material can be obtained.

In one embodiment, the Chinese herbal compound extract in thecomposition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases of the present disclosuremay be obtained by a method. The method mentioned above may comprise thefollowing steps, but it is not limited thereto.

First, the respective herbal materials contained in a preparation rawmaterial for forming the preceding Chinese herbal compound extract arerespectively subjected an extraction procedure to obtain respectiveextract solutions of the respective herbal materials. For example,Ganoderma, red jujube, longan and lotus contained in a preparation rawmaterial for forming the preceding Chinese herbal compound extract arerespectively subjected an extraction procedure with a solvent to obtainrespective Ganoderma extract, red jujube extract, longan extract andlotus seed extract.

Next, the obtained extracts of the respective herbal material are mixedto form a mixture extract to obtain the Chinese herbal compound extractmentioned above. For example, the obtained respective Ganoderma extract,red jujube extract, longan extract and lotus seed extract were mixed toform a mixture extract to obtain the Chinese herbal compound extractmentioned above.

The solvents used in the respective extraction procedures for therespective herbal materials in the preparation raw material can be thesame or different, as long as in the subsequent steps of mixing therespective extract solutions, the solvents used in the respectiveextraction procedures do not affect each other, and do not affect theingredients in the mixture extract. In one specific embodiment, thesolvents used in the respective extraction procedures for the respectiveherbal materials in the preparation raw material all can be water.

The temperatures for the respective extraction procedures for therespective herbal materials in the preparation raw material can be thesame or different, are not particularly limited, and can be adjusted asneeded. For example, the temperatures for the respective extractionprocedures for the respective herbal materials can be adjusted accordingto the environment (such as environmental temperature, humidity, andpressure) at the time at which the extraction procedure is performed,the kind of herbal material to be extracted, the condition of the herbalmaterial (such as moisture content, and weight) to be extracted, theextraction time to be performed, and/or the kind of extraction solventto be used, but they are not limited thereto. For example, thetemperatures for the respective extraction procedures for the respectiveherbal materials in the preparation raw material are above the freezingpoint and below the boiling point of the solvents used in the respectiveextraction procedures. In one embodiment, the solvents used in therespective extraction procedures for the respective herbal materials inthe preparation raw material all are water, and the temperatures for therespective extraction procedures for the respective herbal materials inthe preparation raw material may be about 0-100° C., such as about 4°C., about 5° C., about 10° C., about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., about 37° C., about 40° C., about 50° C.,about 60° C., about 70° C., about 80° C., about 90° C., and about 100°C., but it is not limited thereto.

Similarly, the time for performing the respective extraction proceduresfor the respective herbal materials in the preparation raw material canbe the same or different, is not particularly limited, and can beadjusted as needed. For example, the time for the respective extractionprocedures for the respective herbal materials can be adjusted accordingto the environment (such as environmental temperature, humidity, andpressure) at the time at which the extraction procedure is performed,the kind of herbal material to be extracted, the condition of the herbalmaterial (such as moisture content, and weight) to be extracted, theextraction temperature to be adopted, and/or the kind of extractionsolvent to be used, but it is not limited thereto. For example, the timefor performing the respective extraction procedures for the respectiveherbal materials in the preparation raw material may be about 0.1-10hours, such as about 0.1 hour, about 0.5 hour, about 1 hour, about 1.5hours, about 2 hours, about 2.5 hours, about 5 hours, about 8 hours, andabout 10 hours, but it is not limited thereto. In one embodiment, thesolvents used in the respective extraction procedures for the respectiveherbal materials in the preparation raw material all are water, and thetime for performing the respective extraction procedures for therespective herbal materials in the preparation raw material may be about0.1-10 hours, such as about 0.1 hour, about 0.5 hour, about 1 hour,about 1.5 hours, about 2 hours, about 2.5 hours, about 5 hours, about 8hours, and about 10 hours, but it is not limited thereto.

In addition, according to needs, after the step of mixing the obtainedextracts of respective herbal material to form a mixture extract, theabove-mentioned method for obtaining the Chinese herbal compound extractin the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure may further comprise a step of drying the mixture extractmentioned above. The method for drying is not particularly limited, aslong as the extract can be dried, such as oven drying, and freezedrying. In one embodiment, the mixture extract is dried by freezedrying.

Furthermore, in another embodiment, the Chinese herbal compound extractin the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure may be obtained by another method. The method mentioned abovemay comprise the following steps, but it is not limited thereto.

First, the respective herbal materials contained in a preparation rawmaterial for forming the preceding Chinese herbal compound extract aremixed to form a raw material mixture. For example, Ganoderma, redjujube, longan and lotus seed contained in a preparation raw materialfor forming the preceding Chinese herbal compound extract are mixed toform a raw material mixture.

Next, the foregoing raw material mixture is subjected an extractionprocedure to obtain a mixture extract. For example, a raw materialmixture containing Ganoderma, red jujube, longan and lotus seed issubjected an extraction procedure to obtain a mixture extract to obtainthe Chinese herbal compound extract mentioned above.

The solvent used in the extraction procedure for the raw materialmixture is not particularly limited, and can be adjusted as needed, aslong as it has no adverse effect on the ingredients in the extract. Inone specific embodiment, the solvent used in the extraction procedurefor the raw material mixture can be water.

The temperature for the extraction procedure for the raw materialmixture is not particularly limited, and can be adjusted as needed. Forexample, the temperature for the extraction procedure for the rawmaterial mixture can be adjusted according to the environment (such asenvironmental temperature, humidity, and pressure) at the time at whichthe extraction procedure is performed, the kinds of herbal materialscontained in the raw material mixture to be extracted, the condition ofthe raw material mixture (such as moisture content, and weight) to beextracted, the extraction time to be performed, and/or the kind ofextraction solvent to be used, but it is not limited thereto. Forexample, the temperature for the extraction procedure for the rawmaterial mixture is above the freezing point and below the boiling pointof the solvent used in the extraction procedure. In one embodiment, thesolvent used in the extraction procedure for the raw material mixture iswater, and the temperature for the extraction procedure for the rawmaterial mixture may be about 0-100° C., such as about 4° C., about 5°C., about 10° C., about 15° C., about 20° C., about 25° C., about 30°C., about 35° C., about 37° C., about 40° C., about 50° C., about 60°C., about 70° C., about 80° C., about 90° C., and about 100° C., but itis not limited thereto.

Similarly, the time for performing the extraction procedure for the rawmaterial mixture is not particularly limited, and can be adjusted asneeded. For example, the time for performing the extraction procedurefor the raw material mixture can be adjusted according to theenvironment (such as environmental temperature, humidity, and pressure)at the time at which the extraction procedure is performed, the kinds ofherbal materials contained in the raw material mixture to be extracted,the condition of the raw material mixture (such as moisture content, andweight) to be extracted, the extraction temperature to be adopted,and/or the kind of extraction solvent to be used, but it is not limitedthereto. For example, the time for performing the extraction procedurefor the raw material mixture may be about 0.1-10 hours, such as about0.1 hour, about 0.5 hour, about 1 hour, about 1.5 hours, about 2 hours,about 2.5 hours, about 5 hours, about 8 hours, and about 10 hours, butit is not limited thereto. In one embodiment, the solvent used in theextraction procedure for the raw material mixture is water, and the timefor performing the extraction procedure for the raw material mixture maybe about 0.1-10 hours, such as about 0.1 hour, about 0.5 hour, about 1hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 5 hours,about 8 hours, and about 10 hours, but it is not limited thereto.

In addition, according to needs, after the step of mixing the obtainedextracts of respective herbal material to form a mixture extract, theabove-mentioned method for obtaining the Chinese herbal compound extractin the composition for modulating intestinal permeability and/ortreating and/or preventing leaky gut related diseases of the presentdisclosure may further comprise a step of drying the mixture extractmentioned above. The method for drying is not particularly limited, aslong as the extract can be dried, such as oven drying, and freezedrying. In one embodiment, the mixture extract is dried by freezedrying.

In one embodiment, the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure mentioned above, in addition to theforegoing Chinese herbal compound material or the Chinese herbalcompound extract, may further comprise a pharmaceutically acceptablecarrier or salt, but it is not limited thereto. In this embodiment, inthe composition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases of the present disclosurementioned above, the content of the foregoing Chinese herbal compoundmaterial or the Chinese herbal compound extract may be about 10-99.5 wt%, such as about 10-50 wt %, and about 50-99.5 wt %, but it is notlimited thereto. In this embodiment, the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure mentioned above may be apharmaceutical composition or a health care composition, but it is notlimited thereto.

The pharmaceutically acceptable carrier mentioned above may comprise,but is not limited to, a solvent, a dispersion medium, a coating, anantibacterial and antifungal agent, or an isotonic and absorptiondelaying agent, etc. which is suitable for pharmaceuticaladministration. The pharmaceutical composition can be formulated intodosage forms for different administration routes utilizing conventionalmethods.

Moreover, the pharmaceutically acceptable salt mentioned above maycomprise, but is not limited to, salts including inorganic cation, suchas alkali metal salts such as sodium salt, potassium salt or amine salt,such as alkaline-earth metal salt such as magnesium salt or calciumsalt, such as the salt containing bivalent or quadrivalent cation suchas zinc salt, aluminum salt or zirconium salt. Furthermore, thepharmaceutically acceptable salt may also be organic salt, such asdicyclohexylamine salt, methyl-D-glucamine, and amino acid salt such asarginine, lysine, histidine, or glutamine.

Furthermore, example for the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure mentioned above may comprise, but isnot limited to, a pharmaceutical composition or a health carecomposition, but it is not limited thereto.

The pharmaceutical composition or a health care composition of thepresent disclosure may be administered parenterally, orally, by aninhalation spray, or via an implanted reservoir. The parenteral methodsmay comprise smearing on skin on any region or a region needed thereof,subcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intra-arterial, intrasynovial, intrasternal,intrathecal, intralesional injection, as well as infusion techniques.

The oral form of the pharmaceutical composition or health carecomposition mentioned in the present disclosure may comprise tablets,granules, powders, pellet in capsules, capsules, coated tablets,emulsions, solutions, aqueous suspensions, dispersions, instant powders,etc., but it is not limited thereto.

In one specific embodiment, the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure mentioned above may be apharmaceutical composition. In this specific embodiment, thepharmaceutical composition may be an oral dosage form, wherein the oraldosage form may comprise tablets, granules, powders, pellet in capsules,capsules, coated tablets, emulsions, solutions, aqueous suspensions,dispersions, etc., but it is not limited thereto. Moreover, in thisspecific embodiment, the pharmaceutical composition may comprise, but isnot limited to a pharmaceutical composition for treating and/orpreventing inflammatory bowel diseases, neurodegenerative disordersand/or sleep disorder.

In another specific embodiment, the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases of the present disclosure mentioned above may be ahealth care composition. In this specific embodiment, the health carecomposition may be a health food, wherein the form of the health foodmay comprise tablets, granules, powders, pellet in capsules, capsules,coated tablets, emulsions, solutions, aqueous suspensions, dispersions,instant powders, etc., but it is not limited thereto. Moreover, in thisspecific embodiment, the health care composition may comprise, but isnot limited to, a health care composition for preventing and/orameliorating inflammatory bowel diseases, neurodegenerative disordersand/or sleep disorder.

Based on the foregoing, the present disclosure may further provide a useof a Chinese herbal compound material or a Chinese herbal compoundextract in the manufacture of a composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases.

Moreover, the present disclosure can further provide a use of a Chineseherbal compound material or a Chinese herbal compound extract in themanufacture of a composition for treating and/or preventing inflammatorybowel diseases. The inflammatory bowel disease mentioned herein maycomprise, but is not limited to, colitis, etc. In one embodiment, theinflammatory bowel disease mentioned herein is colitis.

Furthermore, the present disclosure may further provide a use of aChinese herbal compound material or a Chinese herbal compound extract inthe manufacture of a composition for treating and/or preventingneurodegenerative disorders. The neurodegenerative disorder mentionedherein may comprise, but is not limited to, Alzheimer's disease,Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosisor combinations thereof, etc. In one embodiment, the inflammatory boweldisease mentioned herein is Alzheimer's disease.

The present disclosure may yet further provide a use of a Chinese herbalcompound material or a Chinese herbal compound extract in themanufacture of a composition for treating and/or preventing sleepdisorders. The sleep disorder mentioned herein may comprise, but is notlimited to, chronic sleep fragmentation, insomnia or a combinationthereof, etc. In one embodiment, the sleep disorder mentioned herein isinsomnia.

In the respective uses of a Chinese herbal compound material or aChinese herbal compound extract of the present disclosure mentionedabove, the foregoing Chinese herbal compound material or the foregoingChinese herbal compound extract mentioned above has a modulating effecton intestinal permeability and/or has a treating and/or preventingeffect on leaky gut related diseases.

Furthermore, all related interpretations for the Chinese herbal compoundmaterial or the Chinese herbal compound extract involved in therespective uses of the present disclosure mentioned above can bereferred to the description related to the Chinese herbal compoundmaterial or the Chinese herbal compound extract in the proceedingparagraphs for interpreting the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure, and thus are not repeated herein.

In one embodiment, in the respective uses of a Chinese herbal compoundmaterial or a Chinese herbal compound extract of the present disclosurementioned above, a pharmaceutically acceptable carrier or salt may befurther used in a preparation for the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated diseases.

Related interpretations for the pharmaceutically acceptable carrier orsalt can be also referred to the related description in the proceedingparagraphs for interpreting the composition for modulating intestinalpermeability and/or treating and/or preventing leaky gut relateddiseases of the present disclosure, and thus are not repeated herein.

Furthermore, all related interpretations for the compositionmanufactured in the respective uses of the present disclosure can bereferred to all description in the proceeding paragraphs forinterpreting the composition for modulating intestinal permeabilityand/or treating and/or preventing leaky gut related diseases of thepresent disclosure, but it is not limited thereto.

In addition, based on the foregoing, the present disclosure may furtherprovide a method for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases. The method mentioned abovemay comprise, but is not limited to, administering any composition formodulating intestinal permeability and/or treating and/or preventingleaky gut related diseases of the present disclosure mentioned above toa subject in need thereof.

Moreover, based on the foregoing, the present disclosure may furtherprovide a method for treating and/or preventing inflammatory boweldiseases. The method mentioned above may comprise, but is not limitedto, administering any composition for modulating intestinal permeabilityand/or treating and/or preventing leaky gut related diseases of thepresent disclosure mentioned above to a subject in need thereof. Theinflammatory bowel disease mentioned herein may comprise, but is notlimited to, colitis, etc.

Based on the foregoing, the present disclosure may also provide a methodfor treating and/or preventing neurodegenerative disorders. The methodmentioned above may comprise, but is not limited to, administering anycomposition for modulating intestinal permeability and/or treatingand/or preventing leaky gut related diseases of the present disclosurementioned above to a subject in need thereof. The neurodegenerativedisorder mentioned herein may comprise, but is not limited to,Alzheimer's disease, Parkinson's disease, multiple sclerosis,amyotrophic lateral sclerosis or combinations thereof, etc.

Based on the foregoing, the present disclosure may also provide a methodfor treating and/or preventing sleep disorders. The method mentionedabove may comprise, but is not limited to, administering any compositionfor modulating intestinal permeability and/or treating and/or preventingleaky gut related diseases of the present disclosure mentioned above toa subject in need thereof. The sleep disorder mentioned herein maycomprise chronic sleep fragmentation, insomnia or a combination thereof,etc., but it is not limited thereto.

The subject mentioned in the present disclosure may comprise, but is notlimited to, a vertebrate. The vertebrate mentioned above may comprise afish, an amphibian, a reptile, a bird or a mammal, but it is not limitedthereto. Example of the mammal may comprise, but is not limited to ahuman, an orangutan, a monkey, a horse, a donkey, a dog, a cat, arabbit, a guinea pig, a rat, a mouse, etc. In one embodiment, the saidsubject may be a human.

EXAMPLES Example 1: Preparation of Test Samples

40 g of Ganoderma lucidum, 40 g of grey jujube, 40 g of Fen Ke longanand 40 g of red lotus seed were refluxed and extracted with 400 g ofwater for 2 hours, respectively, to obtain a Ganoderma lucidum extractsolution (i) (241 g), a red jujube extract solution (ii) (338 g), alongan extract solution (iii) (286 g) and a lotus seed extract solution(iv) (313 g).

The extracts of the respective herbal materials were taken by the weightcorresponding to the contents (proportions) of the respective herbalmaterials in the preparation raw material in the condition of that thetotal weight of the respective herbal materials in the preparation rawmaterials was set to 1 g, and mixed to prepare 16 test samples shown inthe following Table 1.

TABLE 1 Weight ratio of herbal materials contained in the Sample Herbalmaterial contained in the preparation raw number preparation rawmaterial material  1 Ganoderma —  2 Red jujube —  3 Longan —  4 Lotusseed —  5 Ganoderma and red jujube 1:1  6 Ganoderma and longan 1:1  7Ganoderma and lotus seed 1:1  8 Red jujube and longan 1:1  9 Red jujubeand lotus seed 1:1 10 Longan and lotus seed 1:1 11 Ganoderma, red jujubeand longan 1:1:1 12 Ganoderma, red jujube, and lotus 1:1:1 seed 13Ganoderma, longan and lotus seed 1:1:1 14 Red jujube, longan and lotusseed 1:1:1 15A Ganoderma, red jujube, longan and 1:1:1:1 lotus seed 15BGanoderma, red jujube, longan and 3:1:1:1 lotus seed 15C Ganoderma, redjujube, longan and 0.5:1:1:1 lotus seed 15D Ganoderma, red jujube,longan and 6:1:1:1 lotus seed 15E Ganoderma, red jujube, longan and10:1:1:1 lotus seed 15F Ganoderma, red jujube, longan and 1:0.5:1:1lotus seed 15G Ganoderma, red jujube, longan and 1:3:1:1 lotus seed 15HGanoderma, red jujube, longan and 1:6:1:1 lotus seed 15I Ganoderma, redjujube, longan and 1:10:1:1 lotus seed 15J Ganoderma, red jujube, longanand 1:1:0.5:1 lotus seed 15K Ganoderma, red jujube, longan and 1:1:3:1lotus seed 15L Ganoderma, red jujube, longan and 1:1:6:1 lotus seed 15MGanoderma, red jujube, longan and 1:1:10:1 lotus seed 15N Ganoderma, redjujube, longan and 1:1:1:0.5 lotus seed 15O Ganoderma, red jujube,longan and 1:1:1:3 lotus seed 15P Ganoderma, red jujube, longan and1:1:1:6 lotus seed 15Q Ganoderma, red jujube, longan and 1:1:1:10 lotusseed

For example, for a single-ingredient sample of Ganoderma lucidum,namely, the Ganoderma lucidum extract solution (i) obtained in theforegoing was taken by an amount of 1/40 weight thereof (241 g*(1/40)=6.025 g) (corresponding to 1 g of herbal material of Ganodermalucidum) and freeze dried.

For example, for a combination sample with two-ingredient compound ofGanoderma lucidum and red jujube, namely, the Ganoderma lucidum extractsolution (i) obtained in the foregoing was taken by an amount of 1/80weight thereof (241 g*( 1/80)=3.013 g) and the red jujube extractsolution (ii) obtained in the foregoing was taken by an amount of 1/80weight thereof (338 g*( 1/80)=4.225 g) (corresponding to 0.5 g herbalmaterial of Ganoderma lucidum and 0.5 g herbal material of red jujube,the total weight of the respective herbal materials was 1 g), and thetwo were well mixed and freeze dried.

For example, for a combination sample with three-ingredient compound ofGanoderma lucidum, red jujube and longan, namely, the Ganoderma lucidumextract solution (i) obtained in the foregoing was taken by an amount of1/120 weight thereof (241 g*( 1/120)=2.008 g), the red jujube extractsolution (ii) obtained in the foregoing was taken by an amount of 1/120weight thereof (338 g*( 1/120)=2.817 g) and the longan extract solution(iii) obtained in the foregoing was taken by an amount of 1/120 weightthereof (286 g*( 1/120)=2.383 g) (corresponding to 0.33 g herbalmaterial of Ganoderma lucidum, 0.33 g herbal material of red jujube, and0.33 g herbal material of longan, the total weight of the respectiveherbal material was 1 g), and the three were well mixed and freezedried.

For a combination sample with four-ingredient compound of Ganodermalucidum, red jujube, longan and lotus seed (1:1:1:1), namely, theGanoderma lucidum extract solution (i) obtained in the foregoing wastaken by an amount of 1/160 weight thereof (241 g*( 1/160)=1.506 g), thered jujube extract solution (ii) obtained in the foregoing was taken byan amount of 1/160 weight thereof (338 g*( 1/160)=2.113 g), the longanextract solution (iii) obtained in the foregoing was taken by an amountof 1/160 weight thereof (286 g*( 1/160)=1.788 g) and the lotus seedextract solution (iv) obtained in the foregoing was taken by an amountof 1/160 weight thereof (313 g*( 1/160)=1.956 g) (corresponding to 0.25g herbal material of Ganoderma lucidum, 0.25 g herbal material of redjujube, 0.25 g herbal material of longan and 0.25 g herbal material oflotus seed, the total weight of the respective herbal material was 1 g).

The 16 test samples shown in Table 1 were prepared according to therules described above.

The dry weight of the respective medicinal extracts contained in therespective test samples can be inferred, for example, according to theestimation method shown in Table 2-5 below. Moreover, based on the dryweight of the respective extracts corresponding to that is obtained from1 g of the respective herbal materials shown in Tables 2-5 below, theextractabilities for the respective extracts can be calculated by thefollowing formula.Extractability (%)=Dry weight of extract/Weight of the correspondingherbal material*100

Extractabilities for the respective extracts are shown in the following:

Extractability for the Ganoderma extract: 3.71% (0.0371 g/l g*100)

Extractability for the red jujube extract: 60.3% (0.603 g/l g*100)

Extractability for the longan extract: 54.73% (0.5473 g/l g*100)

Extractability for the lotus seed extract: 22.49% (0.225 g/l g*100)

TABLE 2 Illustrative estimation method for dry weights of extracts ofthe respective herbal materials contained in a test sample withtwo-ingredient compound Herbal materials Weight ratio of Estimated dryweight of respective extracts contained Herbal materials containedcontained in the test sample (mg) Sample in preparation in 1 gperparation Dry weight Ganoderma Red jujube Longan Lotus seed number rawmaterial raw material of sample extract extract extract extract 1Ganoderma — 37.1 37.1 — — — 2 Red jujube — 603 — 603 — — 3 Longan —547.3 — — 547.3 — 4 Lotus seed — 224.9 — — — 224.9 6 Ganoderma 1:1 292.237.1*1/2 = — 547.3*1/2 = — and 18.6 273.7 longan Note: Dry weight ofTest ample 6, 292.2 mg

 Estimated dry weight of Ganoderma extract contained therein, 18.6 mg +Estimated dry weight of longan extract contained therein, 273.7 mg

TABLE 3 Illustrative estimation method for dry weights of extracts ofthe respective herbal materials contained in a test sample withthree-ingredient compound Herbal materials Weight ratio of Estimated dryweight of retrospective containted Herbal materials contained extractscontained in the test sample (mg) Sample in preparation in 1 gpreparation Dry weight Ganoderma Red jujube Longan Lotus seed number rawmaterial raw material of sample extract extract extract extract 1Ganoderma — 37.1 37.1 — — — 2 Red jujube — 603 — 603 — — 3 Longan —547.3 — — 547.3 — 4 Lotus seed — 244.9 — — — 224.9 11 Ganoderma, 1:1:1395.8 37.1*1/3 = 603*1/3 = 547.3*1/3 = — red jujube and 12.4 201 182.4longan Note: Dry weight of Test sample 11, 395.8 mg

 Estimated dry weight of Ganoderma extract contained therein, 12.4 mg +Estimated dry weight of red jujube extract contained therein, 201 mg +Estimated dry weight of longan extract contained therein, 182.4 mg

TABLE 4 Illustrative estimation method for dry weights of extracts ofthe respective herbal materials contained in a test sample withfour-ingredient compound Herbal materials Weight ratio of Estimated dryweight of respective extracts containted Herbal materials containedcontained in the test sample (mg) Sample in preparation in 1 gpreparation Dry weight Ganoderma Red jujube Longan Lotus seed number rawmaterial raw material of sample extract extract extract extract  1Ganoderma — 37.1 37.1 — — —  2 Red jujube — 603 — 603 — —  3 Longan —547.3 — — 547.3 —  4 Lotus seed — 244.9 — — — 224.9 15A Ganoderma,1:1:1:1 352.4 37.1*1/4 = 603*1/4 = 547.3*1/4 = 224.9*1/4 = red jujube,9.3 150.1 136.8 56.2 longan and lotus seed Note: Dry weight of Testsample 15A, 352.4 mg

 Estimated dry weight of Ganoderma extract contained therein, 9.3 mg +Estimated dry weight of red jujube extract contained therein, 150.1 mg +Estimated dry weight of longan extract contained therein, 136.8 mg +Estimated dry weight of lotus seed extract contained therein, 56.2 mg

TABLE 5 Illustrative estimation method for dry weights of extracts ofthe respective herbal materials contained in a test sample withfour-ingredient compound Herbal materials Weight ratio of Estimated dryweight of respective extracts containted Herbal materials containedcontained in the test sample (mg) Sample in preparation in 1 gpreparation Dry weight Ganoderma Red jujube Longan Lotus seed number rawmaterial raw material of sample extract extract extract extract  1Ganoderma — 37.1 37.1 — — —  2 Red jujube — 603 — 603 — —  3 Longan —547.3 — — 547.3 —  4 Lotus seed — 244.9 — — — 224.9 15B Ganoderma, red3:1:1:1 247.8 37.1*3/6 = 603*1/6 = 547.3*1/6 = 224.9*1/6 = jujube,longan 18.6 100.5 91.2 37.5 and lotus seed Note: Dry weight of Testsample 15B, 247.8 mg

 Estimated dry weight of Ganoderma extract contained therein, 18.6 mg +Estimated dry weight of red jujube extract contained therein, 100.5 mg +Estimated dry weight of longan extract contained therein, 91.2 mg +Estimated dry weight of lotus seed extract contained therein, 37.5 mg

Example 2

A. Methods

The 16 test samples prepared above were subjected to a cell lethalconcentration test.

The cell lethal concentration test for the test samples were performedvia the human colon adenocarcinoma cell Caco-2.

The cells were divided into a negative control (NC) and 16 experimentalgroups in which different test samples were used. For the negativecontrol group, untreated cells were used, and for each experimentalgroup, the test sample was diluted from the highest concentration of 200μg/mL to each test concentration by a 2-fold dilution method (first, thetest sample was prepared at a concentration of 100 mg/mL with ddH₂O,diluted with cell culture medium to a concentration of 200 μg/mL, andthen diluted to each test concentration), and the test samples atdifferent concentrations were respectively co-cultured with human colonadenocarcinoma cells Caco-2 in a 96-well culture plate for 48 hours.

After that, 0.5 mg/mL MTT was added to the negative control group andeach experimental group and cultured for 4 hours, and then the mediumwas removed, the Formazan blue-violet crystal was dissolved by DMSO, andthe absorbance at 570 nm was measured.

According to the formula shown below, the cell survival rate wasevaluated.Cell survival rate=(Absorbance at 570 nm for the experimentalgroup/Absorbance at 570 nm for the negative control group (NC))×100%.

B. Results

The results are shown in FIGS. 1A to 1P.

Based on FIG. 1A to FIG. 1P, the highest concentration for each samplethat can be used without cell death as shown in Table 6 can be known.

TABLE 6 The highest concentration for each sample that can be usedwithout cell death Weight ratio of herbal Highest materialsconcentration contained in that can be preparation used without SampleHerbal materials contained in raw cell death number preparation rawmaterial material (μg/mL)  2 Red jujube — 100  3 Longan — 100  4 Lotusseed — 100  8 red jujube and longan 1:1 100 10 Longan and lotus seed 1:1100  9 Red jujube and lotus seed 1:1 25 14 Red jujube, longan and lotusseed 1:1:1 25 11 Ganoderma, red jujube and longan 1:1:1 6 13 Ganoderma,longan and lotus seed 1:1:1 6  1 Ganoderma 3  6 Ganoderma and longan 1:13 12 Ganoderma, red jujube and lotus 1:1:1 3 seed 15A Ganoderma, redjujube, longan and 1:1:1:1 3 lotus seed 15B Ganoderma, red jujube,longan and 3:1:1:1 1.5 lotus seed  5 Ganoderma and red jujube 1:1 0.75 7 Ganoderma and lotus seed 1:1 0.75

Example 3: Effect of Single-Ingredient Extract on Intestinal EpithelialCell Permeability

A. Methods

The intestinal permeability system constructed by human intestinalepithelial cells was used to evaluate the effects of the respectiveextracts obtained in Example 1 on intestinal permeability (Pham V T,Seifert N, Richard N, et al. The effects of fermentation products ofprebiotic fibres on gut barrier and immune functions in vitro [publishedcorrection appears in PeerJ. 2018 Aug. 17; 6: Steinert, Robert[corrected to Steinert, Robert E]]. PeerJ. 2018; 6:e5288. Published 2018Aug. 10. doi:10.7717/peerj.5288). The detailed implementation steps areas follows.

After culturing the human colon adenocarcinoma cell Caco-2 on atranswell culture plate for 21 days, the cells were divided into anegative control (NC), a positive control (PC) and 4 experimental groupsto perform an in vitro leaky gut assay. The negative control group wasuntreated cells. The positive control group was cells treated with 50 μMberberine chloride (Valenzano M C, DiGuilio K, Mercado J, Teter M, To J,Ferraro B, et al. (2015) Remodeling of Tight Junctions and Enhancementof Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer byMicronutrients. PLoS ONE 10(7): e0133926.).

The 4 experimental groups were cells respectively treated with Sample 1,Sample 2, Sample 3, and Sample 4 at a concentration that does not causecell death (Sample 1: 3 μg/mL; Sample 2: 100 μg/mL; Sample 3: 100 μg/mL;Sample 4: 100 μg/mL) (first, the sample was prepared at a concentrationof 100 mg/mL with ddH₂O, diluted with cell culture medium to aconcentration of 200 μg/mL, and then diluted to each testconcentration).

The cells of each group were cultured for 48 hours after the treatmentmentioned above, and then the cells were induced with 350 μg/mLrhamnolipids to induce cell permeabilization.

Next, FITC-dextran 4 (FD4) fluorescent dye was added to the inner plateof the transwell culture plate, and after reacting for 4 hours, theliquid in the lower well of the culture plate was aspirated to detectfluorescence intensity thereof (wavelength of excitation light: 485 nm;wavelength of emission light: 538 nm).

According to the formula shown below, the permeability of intestinalcells was evaluated by the fluorescence intensity of the liquid obtainedfrom the lower well.FD4 leakage rate=(FD4 fluorescence value of the test sample/FD4fluorescence value of the negative control group (NC))×100%.

B. Results

The results are shown in FIG. 2 .

FIG. 2 shows that Sample 1 (Ganoderma extract), Sample 2 (red jujubeextract), Sample 3 (longan extract) and Sample 4 (lotus seed extract)all are capable of significantly reducing the FD4 leakage rate, and thisrepresents that Sample 1 to Sample 4 all have the activity of inhibitingintestinal permeability.

Example 4: Effect of Compound Extract on Intestinal Epithelial CellPermeability

A. Methods

The intestinal permeability system constructed by human intestinalepithelial cells was used to evaluate the effects of the respectiveextracts obtained in Example 1 on intestinal permeability. The detailedimplementation steps are as follows.

After culturing the human colon adenocarcinoma cell Caco-2 on atranswell culture plate for 21 days, the cells were divided into anegative control (NC), a positive control (PC) and 9 experimental groupsto perform an in vitro leaky gut assay. The negative control group wasuntreated cells. The positive control group was cells treated with 50 μMberberine chloride. The 9 experimental groups were cells respectivelytreated with 1.5 μg/mL of Sample 1, Sample 2, Sample 3, Sample 4, Sample6, Sample 9, Sample 11, Sample 12 and Sample 15A.

The cells of each group were cultured for 48 hours after the treatmentmentioned above, and then the cells were induced with 350 μg/mLrhamnolipids to induce cell permeabilization.

Next, FITC-dextran 4 (FD4) fluorescent dye was added to the inner plateof the transwell culture plate, and after reacting for 4 hours, theliquid in the lower well of the culture plate was aspirated to detectfluorescence intensity thereof (wavelength of excitation light: 485 nm;wavelength of emission light: 538 nm).

According to the formula shown below, the permeability of intestinalcells was evaluated by the fluorescence intensity of the liquid obtainedfrom the lower well.FD4 leakage rate=(FD4 fluorescence value of the test sample/FD4fluorescence value of the negative control group (NC))×100%.

B. Results

The results are shown in FIG. 3 .

FIG. 3 shows that when the concentration of Sample 2 (red jujubeextract), Sample 3 (longan extract) and Sample 4 (lotus seed extract) isreduced from the 100 μg/mL used in Example 3 to 1.5 μg/mL, Sample 2,Sample 3 and Sample 4 do not significantly reduce the FD4 leakage rate,while only Sample 1 is capable of significantly reducing FD4 leakagerate.

Furthermore, FIG. 3 also shows that for different compound extracts,only Sample 12 (Ganoderma extract+red jujube extract+lotus seed extract(the weight ratio of the respective herbal raw materials used to obtainthe respective extracts is 1:1:1)) and Sample 15A (Ganoderma extract+redjujube extract+lotus seed extract+lotus seed extract (the weight ratioof the respective herbal raw materials used to obtain the respectiveextracts is 1:1:1:1)) are capable of significantly reducing the FD4leakage rate. At the same concentration (1.5 μg/mL), compared to Sample1 and Sample 12, Sample 15A can more significantly inhibit the FD4leakage rate, and this represents that Sample 15A has a better activityin inhibition of intestinal permeability.

Example 5: Effects of Four-Ingredient Compounds in Different Proportionson Intestinal Epithelial Cell Permeability

A. Methods

The intestinal permeability system constructed by human intestinalepithelial cells was used to evaluate the effects of the respectiveextracts obtained in Example 1 on intestinal permeability. The detailedimplementation steps are as follows.

After culturing the human colon adenocarcinoma cell Caco-2 on atranswell culture plate for 21 days, the cells were divided into anegative control (NC), a positive control (PC) and 6 experimental groupsto perform an in vitro leaky gut assay.

In this experiment, the experiment was conducted in 4 batches. In eachbatch, the negative control groups were all untreated cells, and thepositive control groups were all cells treated with 50 μM berberinechloride.

In the first batch, the 6 experimental groups were cells treated with1.5 μg/mL of Sample 1, Sample 15A, Sample 15B, Sample 15C, Sample 15D,and Sample 15E, respectively.

In the second batch, the 6 experimental groups were cells treated with1.5 μg/mL of Sample 1, Sample 15A, Sample 15F, Sample 15G, Sample 15H,and Sample 151, respectively.

In the third batch, the 6 experimental groups were cells treated with1.5 μg/mL of Sample 1, Sample 15A, Sample 15J, Sample 15K, Sample 15L,and Sample 15M, respectively.

In the fourth batch, the 6 experimental groups were cells treated with1.5 μg/mL of Sample 1, Sample 15A, Sample 15N, Sample 150, Sample 15P,and Sample 15Q.

The cells of each group were cultured for 48 hours after the treatmentmentioned above, and then the cells were induced with 350 μg/mLrhamnolipids to induce cell permeabilization.

Next, FITC-dextran 4 (FD4) fluorescent dye was added to the inner plateof the transwell culture plate, and after reacting for 4 hours, theliquid in the lower well of the culture plate was aspirated to detectfluorescence intensity thereof (wavelength of excitation light: 485 nm;wavelength of emission light: 538 nm).

According to the formula shown below, the permeability of intestinalcells was evaluated by the fluorescence intensity of the liquid obtainedfrom the lower well.FD4 leakage rate=(FD4 fluorescence value of the test sample/FD4fluorescence value of the negative control group (NC))×100%.

B. Results

The results of the first batch of experiments, the second batch ofexperiments, the third batch of experiments, and the fourth batch ofexperiments are shown in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D,respectively.

According to FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D, it can be seen thatSample 15A (Ganoderma extract+red jujube extract+lotus seedextract+lotus seed extract (the weight ratio of the respective herbalraw materials used to obtain the respective extracts is 1:1:1:1)) andSample 15B (Ganoderma extract+red jujube extract+lotus seedextract+lotus seed extract (the weight ratio of the respective herbalraw materials used to obtain the respective extracts is 3:1:1:1)) havebetter activities in inhibition of intestinal permeability, whereinSample 15A can inhibit intestinal permeability more significantly thanSample 15B.

Example 6: Effects of Four-Ingredient Compound Extract on IntestinalEpithelial Cell Permeability

The intestinal permeability system constructed by human intestinalepithelial cells was used to evaluate the effect of Sample 15B(Ganoderma extract+red jujube extract+lotus seed extract+lotus seedextract (the weight ratio of the respective herbal raw materials used toobtain the respective extracts is 3:1:1:1)) obtained in Example 1 onintestinal permeability. The detailed implementation steps are asfollows.

After culturing the human colon adenocarcinoma cell Caco-2 on atranswell culture plate for 21 days, the cells were divided into twonegative control (NC) groups, two positive control (PC) groups and 6experimental groups to perform an in vitro leaky gut assay The twonegative control groups were untreated cells. The two positive controlgroups were cells treated with 50 μM berberine chloride. The 6experimental groups were cells respectively treated with Sample 15B atconcentrations of 0.3 μg/mL, 0.3 μg/mL, 0.6 μg/mL, 0.6 μg/mL, 1.5 μg/mL,and 1.5 μg/mL, respectively.

The cells in each group were cultured for 48 hours after the abovetreatment, and then the cells in half of the groups in the experiment(including a negative control group, a positive control group andexperimental groups treated with 3 concentrations of Sample 15B) weretreated at 350 μg/mL rhamnolipids to induce cell permeabilization whilethe cells in the other half of the groups in the experiment were nottreated with rhamnolipid.

Next, FITC-dextran 4 (FD4) fluorescent dye was added to the inner plateof the transwell culture plate, and after reacting for 4 hours, theliquid in the lower well of the culture plate was aspirated to detectfluorescence intensity thereof (wavelength of excitation light: 485 nm;wavelength of emission light: 538 nm).

According to the formula shown below, the permeability of intestinalcells was evaluated by the fluorescence intensity of the liquid obtainedfrom the lower well.FD4 leakage rate=(FD4 fluorescence value of the test sample/FD4fluorescence value of the negative control group (NC))×100%.

B. Results

The results are shown in FIG. 5 . The cells in the groups not treatedwith rhamnolipid showed no obvious fluorescence intensity. In thenegative control groups, compared to the group not induced withrhamnolipid, the fluorescence intensity of the group induced withrhamnolipid was significantly increased, and in the groups induced withrhamnolipid, Sample 15B (Ganoderma extract+red jujube extract+lotus seedextract+lotus seed extract (the weight ratio of the respective herbalraw materials used to obtain the respective extracts is 3:1:1:1)) atconcentrations of 0.6 μg/mL and 1.5 μg/mL could significantly reduce thefluorescence intensity, and this represents that it has an activity ininhibition of intestinal permeability.

Example 7: Effect of Four-Ingredient Compound Extract on Gene ExpressionLevels of Adherent and Tight Junctions Proteins of Intestinal EpithelialCells

A. Methods

The cells in the negative control group, the positive control group andthe experimental group (treated with Sample 15B at a concentration of1.5 μg/mL) in the groups induced by rhamnolipid in Example 6 werecollected.

Total ribonucleic acid (total RNA) of cells was isolated by Total RNAPurification Kit (GeneMark, Taichung, Taiwan). Using Maxima First StrandcDNA Synthesis Kit (Thermo Fisher Scientific), according to themanufacturer's operating procedures, the obtained total ribonucleic acidsubjected to a reverse transcription polymerase chain reaction toreverse-transcript messenger ribonucleic acid (mRNA) into cDNA.

Next, the cDNA samples were subjected to a real-time quantitativepolymerase chain reaction to determine the expression levels of CLDN3,OCLN and TJP1 mRNA. The difference in gene expression was calculated bya relative quantitative data analysis through 2^(−(ΔΔCT)), andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as thereference gene. The primers used for the respective genes are shown inTable 7.

TABLE 7 Primers used for respective genes Gene Forward primer (5′-3′)Reverse primer (5′-3′) CLDN3 ATCGTGTGCTGCGCGTT GGCCCTCCCAGATGTTCTG (SEQID NO. 1) (SEQ ID NO. 2) OCLN GTCCAATATTTTGTGGG GGCACGTCCTGTGTGCCTACAAGG (SEQ ID NO. 4) (SEQ ID NO. 3) TJP1 AGAAGGATGTTTATCGTCCAAGAGCCCAGTTTTCCAT CGCATT (SEQ ID NO. 6) (SEQ ID NO. 5) GAPDHCAAGGTCATCCATGACA GTCCACCACCCTGTTGCTGT ACTTTG AG (SEQ ID NO. 7) (SEQ IDNO. 8)

B. Results

The results are shown in FIGS. 6A, 6B and 6C. The results showed thatcompared to the negative control group, the positive control group andthe experimental group can significantly increase the expression levelsof CLDN3, OCLN and TJP1 genes.

Example 8: Effect of Four-Ingredient Compound Extract on Dextran SulfateSodium Salt (DSS) Induced Abnormal Intestinal Permeability in Animals

A. Methods

The animals used in this experiment were 5-6 week old C57BL/6 male mice(purchased from the National Laboratory Animal Center).

The experiment was performed by an animal model of dextran sulfatesodium salt (DSS) induced abnormal intestinal permeability (Laroui H,Ingersoll S A, Liu H C, Baker M T, Ayyadurai S, et al. (2012) DextranSodium Sulfate (DSS) Induces Colitis in Mice by FormingNano-Lipocomplexes with Medium-Chain-Length Fatty Acids in the Colon.PLoS ONE 7(3): e32084. doi:10.1371/journal.pone.0032084).

The mice were divided into an untreated group (Naive), a negativecontrol group (vehicle: water), a positive control group(5-aminosalicylic acid (5-ASA)) and 3 experiments groups (Sample 15B(Ganoderma extract+red jujube extract+lotus seed extract+lotus seedextract (the weight ratio of the respective herbal raw materials used toobtain the respective extracts is 3:1:1:1))). The mice in the untreatedgroup were not treated in any way. The mice in the negative controlgroup were given drinking water containing 0.5% (v/v) dextran sodiumsulfate (MP Biomedicals) to induce abnormal intestinal permeability; themice in the positive control group were given 0.5% (v/v) dextran sulfatesodium salt and 200 μM 5-aminosalicylic acid. The mice in the 3experimental groups were given 0.5% (v/v) dextran sulfate sodium saltand 100 mg/kg Sample 15B, 0.5% (v/v) dextran sulfate sodium salt and 200mg/kg Sample 15B, and 0.5% (v/v) dextran sulfate sodium salt and 400mg/kg Sample 15B, respectively.

The experiment lasted for 14 days. On the day of the end of theexperiment, after fasting the mice for 3-4 hours, the mice were givenFITC-dextran (FD4, Sigma) at 500 mg/kg. After 2 hours, the mice weresacrificed, their blood was collected, and the serum was separated.

The FITC-dextran content in mouse serum was measured with amultifunctional microplate spectrometer (Molecular Device, FlexStation®3) as an indicator of intestinal permeability.

B. Results

FIG. 7 shows that compared to the untreated group, the concentration ofFITC-FITC-dextran in the serum of the negative control group treatedwith 0.5% (v/v) DSS significantly increases, and this shows that 0.5%(v/v) dextran sulfate sodium salt can induce increase of intestinalpermeability in mice. Moreover, compared to the negative control group,the positive control group and the experimental groups can reduce theserum FITC-dextran concentration, and this shows that Sample 15B(Ganoderma extract+red jujube extract+lotus seed extract+lotus seedextract (the weight ratio of the respective herbal raw materials used toobtain the respective extracts is 3:1:1:1)) can ameliorate the increasein intestinal permeability induced by dextran sulfate sodium salt.

Example 9: The Effect of Four-Ingredient Compound Extract on CircadianDisruption Animal Model

A. Methods

The animals used in this experiment were 8-week-old C57BL/6 male mice(purchased from the National Laboratory Animal Center).

The mice were subjected to a surgical operation to load electrodes onthe skull to record brain waves. After a 7-day recovery period, the micewere reared for 2 weeks under a normal light-dark cycle of 12 hours: 12hours, and the 24-hour brainwave changes of the mice were recorded asthe basic point.

Next, the mice were divided into an untreated group (Naive), a negativecontrol group (vehicle), a positive control group (Diazepam) and anexperimental group (Sample 15B (Ganoderma extract+red jujubeextract+lotus seed extract+lotus seed extract (the weight ratio of therespective herbal raw materials used to obtain the respective extractsis 3:1:1:1)). Mice in the untreated group were kept in a normallight-dark cycle for two weeks (the light-dark cycle was 12 hours: 12hours). The mice in the negative control group, the positive controlgroup and the experimental group were reared for 7 days under thecondition of a light-dark cycle of 7 hours: 7 hours to induce insomniain the mice through circadian disruption. During the feeding, the micein the negative control group were given the same amount of sterilewater, mice in the positive control group were given diazepam (2.5 mg/kgorally), and mice in the experimental group were given 200 mg/kg Sample15B.

The 24-hour brainwave changes of mice were recorded on Day 4 after thecircadian disruption to analyze sleep cycle parameters of the timeproportions (%) of the rapid eye movement (REM) and non-rapid eyemovement (NREM), etc.

B. Results

The results are shown in FIG. 8 . The results show that compared to theuntreated group, the time proportion of the rapid eye movement and thetime proportion of the non-rapid eye movement of the negative controlgroup are both significantly reduced, and this indicates that thecircadian disruption can induce insomnia in mice. Moreover, compared tothe negative control group, both the positive control group and theexperimental group can significantly increase the time proportion of thenon-rapid eye movement, and have the effect of alleviating insomnia inmice.

Example 10: Effect of Four-Ingredient Compound Extract on Amyloidβ-Induced Spatial Learning Disability of Animals

A. Methods

The effect of Sample 15B on learning and operating ability was evaluatedby an animal model in which formation of amyloid-β plaque deposition inanimal brains was induced by amyloid β (Kim, H Y, Lee, D K, Chung, B R,Kim, H V, Kim, Y. Intracerebroventricular Injection of Amyloid-βPeptides in Normal Mice to Acutely Induce Alzheimer-like CognitiveDeficits. J. Vis. Exp. (109), e53308, doi:10.3791/53308 (2016).). Theexperimental steps are described as follows:

The animals used in this experiment were Sprague-Dawley male rats (bodyweight 300-330 g, purchased from Lesco).

The rats were divided into a Sham group, a control group and anexperimental group. The rats in the Sham group were injected withartificial cerebrospinal fluid, the rats in the control group wereinjected with 2.5 μL amyloid β peptide 1-40, and the rats in theexperimental group were injected with 2.5 μL amyloid β peptide 1-40 andorally administered with Sample 15B (127 mg/kg/day) for 30 days,continuously.

Evaluation tests of locomotion and exploratory behavior were conductedon Day 22 to Day 23 of the experiment

The Evaluation for locomotion and exploratory behavior are described asfollows:

Evaluation tests of locomotion and exploratory behavior for rats wereperformed in an experiment box. The experiment box had a size of 40 cmin length, width and height. In addition, the experiment box had astainless steel bottom plate, and the bottom plate had 16 holes with adiameter of 3 cm, which were arranged in a 4*4 matrix, and the distancebetween two adjacent holes was 4 cm, and the distance between the eachside of the matrix and the hole was 7 cm. The movement of the rat wassensed and record by the sensors, TruScan Line E63-01HS and TruScanSensor E63-22 (Coulbourn Instruments International Corporation), andanalyzed with the analysis software, Coulbourn Instruments' The HabitestSystem. The measurement time for each rat was 15 minutes. Themeasurement included the time spent in the holes, the frequency ofhole-poking and the exploratory activities.

A water maze test was performed on the Day 24 to Day 28 of theexperiment.

The water maze test is described as follows:

A swimming pool was divided into four quadrants, and the safe platformwas fixed on the fourth quadrant. The rats were placed in the swimmingpool (the respective rats were placed in a different quadrant in eachtest) and trained twice a day for 2 minutes each time. If a rat found asafe platform within 2 minutes, after the rat was allowed to rest for 30seconds, the rat was allowed to leave the swimming pool and rest for 30seconds, and then the next training was proceeded. If the rat had notfound a safe platform within 2 minutes, after the rat was placed on thesafe platform and rest for 30 seconds, the rat was allowed to leave theswimming pool and rest for 30 seconds, and then the next training wasproceeded; The rats were trained for a total of 4 days.

Afterwards, a spatial performance in Morris water maze test and anon-spatial performance in Morris water maze test were performedrespectively. In the spatial performance in Morris water maze test, areference point was established at the relative position to the safeplatform, and then a rat was placed in the first quadrant, and the timerequired for the rat to reach the former safety platform in the swimmingpool was recorded. In the non-spatial performance in Morris water mazetest, the reference point was removed, and a rat was placed in the firstquadrant, and the time required for the rat to reach the former safeplatform in the swimming pool was recorded.

On the next day after the end of the behavioral tests, the hippocampusarea and frontal cortex areas of the brains of all animals were takenout to perform AChE activity analysis and determine the protein content.

B. Results

FIGS. 9A, 9B, and 9C show the results of the evaluation test of thelocomotion and exploratory behavior of rats. The results shows thatcompared to the rats in the Sham group, the time spent in the holes, thenumber of times for hole-poking and the exploration activities for therats in the control group given β-amyloid peptide 1-40 are reduced.However, the experimental group showed that Sample 15B can amelioratethe decrease for the time spent in the holes, the frequency ofhole-poking and the exploration activities of the rats that caused byβ-amyloid peptide 1-40, and can increase the time spent in the holes,the frequency of hole-poking and the exploration activities of the rats.

FIGS. 10 and 11 shows the results of the water maze test in rats.

FIG. 10 shows the time taken for rats to find a safe platform in thespatial performance in Morris water maze test. FIG. 10 shows thatcompared to the rats in the Sham group, the time for the rats in thecontrol group given β-amyloid peptide 1-40 to reach the safety platformof the swimming pool was significantly longer. However, the experimentalgroup showed that Sample 15B can ameliorate the spatial learningdisabilities appears on Day 1 to Day 2, caused by 3-amyloid peptide 1-40in rats (i.e. shorten the time required to reach the safe platform).

FIG. 11 shows the time for rats to reach the safe platform in thenon-spatial performance in Morris water maze test.

Compared to the rats in the Sham group, the rats in the control groupgiven with β-amyloid peptide 1-40 spent significantly longer time tosearch for the area in which the safe platform placed. However, theexperimental group shows that Sample 15B can ameliorate the referencememory difficulties caused by β-amyloid peptide 1-40 in rats (i.e.,shorten the time to search for the area in which the safe platformplaced in the swimming pool).

FIGS. 12A and 12B respectively show the AChE activity in the hippocampusarea of rats and the AChE activity in the frontal cortex area of rats inthe animal model of β-amyloid-induced learning impairment.

FIGS. 12A and 12B show that compared to rats in the Sham group, the AChEactivity in the hippocampus area and frontal cortex area of the controlgroup of rats given β-amyloid peptide 1-40 is significantly greaterincrease. However, the experimental group shows that Sample 15B canameliorate the increase in AChE activity in the hippocampus area andfrontal cortex area of rats that caused by β-amyloid peptide 1-40.

Example 11: Determination of the Content of Ganoderic Acid A in theFour-Ingredient Compound

A. Methods

Ganoderic acid A content determination:

1. Preparation of Reference Standard Stock Solution:

Accurately weighed 10 mg of a reference standard of ganoderic acid A wasplaced in a 10 mL volumetric flask, and then methanol solution was addedthereto, sonication was performed to completely dissolve the ganodericacid A, and then the above solution was quantified to 10 mL to make thesolution contains 1 mg ganoderic acid A per 1 mL to obtain a referencestandard solution (1 mg/mL).

2. Repeatability of Injection:

1 mL of the reference standard solution (1 mg/mL) was taken and placedit in a 10 mL volumetric flask, and then quantify to the graduated markwith methanol. The above solution (0.1 mg/mL) was injected five timesthrough HPLC analysis (Repeatability of injection), and the relativestandard deviation (RSD) of the calculated peak area of ganoderic acid Ashould not be greater than 2.0%.

3. Preparation of Calibration Curve (Ganoderic Acid A Standard):

The stock solution of the reference standard was taken and diluted withmethanol, and based on the sample concentration, ganoderic acid Astandard was prepared for five concentrations, the concentration rangethereof had to include 80-120% of the ganoderic acid A concentration inthe sample, and the ganoderic acid A standard solutions with fiveconcentrations were injected into high performance liquid chromatography(HPLC) for analysis, and the peak area was linearly regressed againstthe concentration, wherein R2 thereof should not be less than 0.995.

4. Test Article Preparation:

1.0 g of the test article (Ganoderma extract (Sample 1) or Sample 15B)was placed in a 20 mL volumetric flask, then about 10 mL of pure wateradded thereto, and the volumetric flask was placed in a water bath forsonication 10 minutes. After standing to cool, pure water was added tothe volumetric flask to quantify to 20 mL to form an inspection samplesolution. An appropriate amount of the inspection sample solution wascentrifuged (10000 rpm, 5 minutes) and filtered with a 0.45 μm filtermembrane, and then high performance liquid chromatography analysis wasperformed to calculate the content of ganoderic acid A in the testarticle.

5. High Performance Liquid Chromatography Analysis Conditions:

Chromatography column type: Inertsil 5 ODS-2 4.6×250 mm or equivalent

Detection wavelength: 257 nm

Pump flow rate: 1.0 mL/minute

Analysis time per sample: 120 minutes

Total injection volume: 20 μL

Mobile Phase Preparation:

Mobile phase A=10% MeOH in CH₃CN (volume ratio)

Mobile phase B=0.075% phosphoric acid aqueous solution (522 μL of 85%phosphoric acid (density=1.685 g/mL) was placed in a 1 L volumetricflask, and diluted with pure water to the graduated mark).

6. Calculation of Ganoderic Acid a ContentThe content of ganoderic acid A (mg/g)=the concentration of ganodericacid A in the inspection sample (mg/mL)×20 (mL)

B. Results

The inspection samples for the ganoderic acid A standard, the Ganodermaextract (Sample 1) and Sample 15B prepared as described above weresubjected to the above-mentioned high performance liquid chromatographyanalysis and the content of ganoderic acid A in the Sample 15B wascalculated. The results are shown in FIG. 13 .

Referring to FIG. 13 , based on the calibration curve of theabove-mentioned standard, the content of ganoderic acid A in theGanoderma extract and Sample 15B can be calculated to be 66.3 mg/g and4.96 mg/g, respectively.

By determining the content of ganoderic acid A in the sample, thequality of the sample can be confirmed.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the disclosed embodiments.It is intended that the specification and examples be considered asexemplary only, with a true scope of the disclosure being indicated bythe following claims and their equivalents.

What is claimed is:
 1. A method for inhibiting intestinal permeability,treating leaky gut related disease and/or preventing leaky gut relateddiseases, comprising: administering a composition for inhibitingintestinal permeability, treating leaky gut related disease and/orpreventing leaky gut related diseases to a subject in need thereof,wherein the composition for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseases,comprises: an herbal compound material or an herbal compound extract,wherein the herbal compound material comprises: Ganoderma; red jujube;longan; and lotus seed, wherein in the herbal compound material, aweight ratio of Ganoderma, red jujube, longan and lotus seed is0.1-15:0.6-2:0.6-2:0.6-5, and wherein the herbal compound extractcomprises: Ganoderma extract; red jujube extract; longan extract; andlotus seed extract, wherein a weight ratio of Ganoderma, red jujube,longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theherbal compound extract is 0.1-15:0.6-2:0.6-2:0.6-5, and wherein theleaky gut related diseases comprise inflammatory bowel disease, celiacdisease, irritable bowel syndrome, acute pancreatitis, non-alcoholicsteatohepatitis, alcoholic cirrhosis, type 1 diabetes, type 2 diabetes,obesity, chronic kidney disease, cardiovascular disease, multiple organfailure syndrome, AIDS, asthma, eczema, psoriasis, autism, depression,anxiety, schizophrenia, bipolar disorder, Alzheimer's disease,Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis,ankylosing spondylitis, fibromyalgia, chronic sleep fragmentation orinsomnia.
 2. The method for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseasesas claimed in claim 1, wherein in the composition for modulatingintestinal permeability and/or treating and/or preventing leaky gutrelated disease, the composition per gram contains 0.2-20 mg ofganoderic acid A.
 3. The method for inhibiting intestinal permeability,treating leaky gut related disease and/or preventing leaky gut relateddiseases as claimed in claim 1, wherein in the herbal compound material,a weight ratio of Ganoderma, red jujube, longan and lotus seed is1:1:1:1.
 4. The method for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseasesas claimed in claim 1, wherein in the herbal compound material, a weightratio of Ganoderma, red jujube, longan and lotus seed is 3:1:1:1.
 5. Themethod for inhibiting intestinal permeability, treating leaky gutrelated disease and/or preventing leaky gut related diseases as claimedin claim 1, wherein a weight ratio of Ganoderma, red jujube, longan andlotus seed as preparation raw materials which are respectively needed toobtain the Ganoderma extract, the red jujube extract, the longan extractand the lotus seed extract contained in the herbal compound extract is1:1:1:1.
 6. The method for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseasesas claimed in claim 1, wherein a weight ratio of Ganoderma, red jujube,longan and lotus seed as preparation raw materials which arerespectively needed to obtain the Ganoderma extract, the red jujubeextract, the longan extract and the lotus seed extract contained in theherbal compound extract is 3:1:1:1.
 7. The method for inhibitingintestinal permeability, treating leaky gut related disease and/orpreventing leaky gut related diseases as claimed in claim 1, wherein apharmaceutically acceptable carrier or salt is further used in apreparation for the composition for inhibiting intestinal permeability,treating leaky gut related disease and/or preventing leaky gut relateddiseases.
 8. The method for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseasesas claimed in claim 7, wherein in the composition for inhibitingintestinal permeability, treating leaky gut related disease and/orpreventing leaky gut related diseases, a content of the herbal compoundmaterial or a herbal compound extract is 10-99.5 wt %.
 9. The method forinhibiting intestinal permeability, treating leaky gut related diseaseand/or preventing leaky gut related diseases as claimed in claim 1,wherein the composition for inhibiting intestinal permeability, treatingleaky gut related disease and/or preventing leaky gut related diseasesis a pharmaceutical composition or a health care composition.